Increased enzymatic activity in rice leaf blast suppression by crude extract of Epicoccum sp

Increased enzymatic activity in rice leaf blast suppression by crude extract of Epicoccum sp.

Ana P.A. Sena^I; Amanda A. Chaibub^I; Márcio V.C.B. Côrtes^II; Gisele B. Silva^III; Valácia L. Silva-Lobo^II; Anne S. Prabhu^II; Marta C.C. Filippi^II; Leila G. Araújo^I

^IDepartamento de Biologia Geral, Universidade Federal de Goiás, Cx. Postal 131, 74001-970, Goiânia, GO, Brazil;

^IIEmbrapa Arroz e Feijão, Cx. Postal 179, 75375-000, Santo Antônio de Goiás, GO, Brazil;

^IIIInstituto de Ciências Agrárias, Universidade Federal Rural da Amazônia, Cx. Postal, 135, 66050-130, Belém, PA, Brasil

^{Correspondence}

ABSTRACT

Epicoccum sp. showed in vitro antagonism to the rice pathogens Magnaporthe oryzae, Rhizoctonia solani, Sarocladium oryzae, Monographella albescens and Cochliobolus miyabeanus in dual culture. The colony growth of the rice blast fungus, M. oryzae, was reduced by 42.50%. The lethal doses (LD₅₀) determined based on probit-log dosage response curves at 3 and 6 hours after germination were 393.0 and 326.6 ppm, respectively. The crude extract (600 ppm) reduced appressorial formation by 95.68%. A greenhouse experiment comparing the relative efficiency of conidial suspension and crude extract of Epicoccum sp. in the suppression of leaf blast showed no statistical difference between both application methods. However, the crude extract of Epicoccum sp. (4000 ppm) 48 hours before the application of M. oryzae induced resistance and suppressed leaf blast by 97.6%. Scanning electron microscopy of rice leaves inoculated with crude extract of Epicoccum sp. and challenged with M. oryzae showed appressorial deformation on penetration phase. Peroxidase and β-1,3-glucanase activities increased in plants sprayed with crude extract of Epicoccum sp., 24 hours after the application of the challenger. PAL as well as chitinase activities increased 72 hours after challenge inoculation. Epicoccum sp. was shown to be a potential antagonist and inducer of resistance against M. oryzae.

Key words:Epicoccum sp., Magnaporthe oryzae, Oryza sativa, antagonism, ISR, PR proteins.

INTRODUCTION

Brazil is the major upland rice producer in the world. This cereal is very important because more than half of the world population depends on rice as a staple food (FAO, 2011). Rice blast [Magnaporthe oryzae B. Couch, anamorph Pyricularia oryzae Cavara] is reported in all rice growing regions in the world and grain yield losses due to this disease may reach up to 100% (Prabhu et al., 2009; Fisher et al., 2012). Under tropical climatic conditions, combined with pathogen biology and limited durability of cultivar resistance, growers resort to intensive application of agricultural chemicals which result in environmental damage such as soil and water contamination, destruction of non-target microorganisms, human health hazards, selection of new pathotypes, besides increased production costs (Ramos, 2009).

Currently, plant disease biocontrol is considered economically viable because it reduces the negative impact on the environment and the risk of altering food integrity when compared with chemical control. Therefore, research on biological control is currently receiving greater attention as it is being considered as one of the main components to be included in integrated disease management for sustainable agriculture (Lahlali & Hijri, 2010). According to the model proposed by McSpadden-Gardener & Fravel (2002), the first line of defense in disease and pest management includes good agricultural practices, whereas the use of resistant cultivars developed by either traditional breeding or genetic engineering constitutes the second line of defense. However, these complementary measures for promoting economically sustainable agriculture must include biocontrol practices for diseases (Pal & McSpadden-Gardener, 2006).

Among the biological control agents, the fungus Epicoccum sp. of the order Moniliales and family Dematiaceae (Barnett & Hunter, 2006) is registered as an antagonist for Monilinia sp. in peach and nectarine (De Cal et al., 2009), Sclerotinia sclerotiorum in sunflower and dry beans (Huang et al., 2000; Pieckenstain et al., 2001), Monilinia fructicola in plums and apples (Wittig et al., 1997; Falconi & Mendgen, 1994), Erysiphe cichoracearum in lady's fingers (Derbalah et al., 2011), Colletotrichum gloesporioides in guava (Pandey et al., 2007), Rhizoctonia solani in tomato (Lahlali & Hijri, 2010), Macrophomina phaseolina and R. solani in soybean (Hashem, 2004), R. solani and Fusarium sambuciunum in potato (El-Kot, 2008). Also, its efficiency as antagonist to Oomycetes such as Pythium in Vicia faba, Vicia unguiculata, Lupinus termis (Koutb & Ali, 2010), Gossypium hirsutum (Hashem & Ali, 2004), Plasmopora viticola in Vitis vinifera (Kortekamp, 1997), and phytoplasm in Catharanthus roseus (Musetti et al., 2011) has been reported. Out of 189 fungal isolates obtained from rice phylloplane, 17 fungi were identified of which Epicoccum sp. (EP06) was the most efficient as an antagonist (Sena, 2012).

These biocontrol agents can act directly or indirectly by induction of defense mechanisms in plant diseases control (Doohan, 2005). The use of biotic and abiotic resistance inducers is one of the major strategies for increasing durability of disease resistance and to reduce toxic residues produced by the indiscriminate use of agricultural chemicals (Filippi et al., 2011). Studies on induction of acquired systemic resistance (SAR) and induced systemic resistance (ISR) in rice plants to M. oryzae were conducted after the application of avirulent isolates of M. oryzae, Bipolaris sorokiniana and rhizobacteria (Smith & Métraux 1991; Manandhar et al., 1998; Tsukamoto et al., 1999; Ashizawa et al., 2005; Filippi et al., 2007, 2011).

During SAR and ISR pathways key enzymes are expressed which are related to the metabolism of phenylpropanoids, such as phenylalanine ammonia lyase, peroxidases, polyphenoloxidases and pathogenesis-related (PR) proteins (Yoshikawa et al., 1983). Some examples of PR proteins related to SAR are β-1.3-glucanase and chitinase, beloning to the PR-2 and PR-3 families, respectively (Van Loon and Pieterse, 2006). However, the duration of protection by SAR is shorter compared to the protection mediated by agents of ISR (Wei et al., 1991).

Studies on the interaction between M. oryzae in rice and Epicoccum sp., and the mechanisms of in vivo biological control are important for the adoption of adequate control measures for rice blast which are ecologically sustainable (Filippi et al., 2007). The knowledge of the mode of action and mechanisms involved in disease reduction by potential biocontrol agents such as Epicoccum sp. is limited. The present study reports the potential of crude extract of Epicoccum sp. in leaf blast suppression and elucidates the involved mechanisms on the basis of enzymatic activity.

MATERIAL AND METHODS

Antagonism of Epicoccum sp. against rice pathogens

Epicoccum sp. was isolated by Sena (2012) from the phylloplane of BRS Cateto rice cultivar from a comercial organic field, at Hidrolândia, GO. The following rice pathogens were used in antibiosis tests: M. oryzae; R. solani (sheath blight); Sarocladium oryzae (sheath rot); Monographella albescens (leaf scald) and Cochliobolus miyabeanus (brown spot), all obtained from the culture collection of Embrapa Arroz e Feijão. These isolates as well as the Epicoccum sp. isolate EP06 were transferred to Petri plates containing potato-dextrose-agar (PDA) and maintained at 25°C for four days under fluorescent light to obtain mycelial discs for antibiosis test. The experiment was conducted utilizing the dual culture technique (Romeiro, 2007). Mycelial discs 5 mm in size of each pathogen and Epicoccum sp. were placed in opposite sides of a plate containing PDA, maintaining a distance of 3.0 cm. The bioassay was run in a completely randomized design with three replications. Three plates for each pathogen without antagonist were used as control.

The evaluations were made 12 days after incubation under continuous fluorescent light at 25°C, measuring the colony diameter in horizontal and vertical directions of the pathogens and the isolate of Epicoccum sp. Also, the reduction in radial growth (the horizontal colony diameter times the vertical colony diameter) was calculated as (mean value of radial growth of the pathogen in the presence of the antagonist x 100/radial growth of control)-100.

Effect of crude extract of Epicoccum sp. on conidial germination and appressorial formation by M. oryzae

Crude extract preparation

The isolate of Epicoccum sp. was multiplied on PDA and after 18 days the colonies were cut and ground in a beaker with 100 mL of ethanol 95%. The suspension was filtered using a funnel containing filter paper (Whatman no. 1) for recovering part of the liquid in a Falcon tube. The liquid was centrifuged for 10 minutes at 500 rpm and the supernatant was concentrated by evaporation under low pressure utilizing a roto-vaporizer II at 40°C under vacuum, and later submitted to lyophilization for obtaining crystals of Epicoccum sp.

Preparation of conidial suspension of M. oryzae and Epicoccum sp.

The isolate of M. oryzae (Py 4075), showing compatible reaction to the cultivar Primavera, obtained from the Microorganism Multifunction Collection of Embrapa Arroz e Feijão, was multiplied in Petri dishes containing oat meal-dextrose-agar and incubated under continuous fluorescent light at 25-27°C for mycelial growth. After 14 days of incubation, the pathogen was subjected to physical stress by scraping the mycelium with a sterilized glass rod. The plates were transferred to a growth chamber at 25-27°C and were covered with a transparent plastic sheet under continuous fluorescent light for 48 to 72 hours. The inoculum was prepared by flooding the plates with sterile distilled water and removing the conidia with a paint brush. The conidial suspension was filtered through a sterilized cheesecloth and the concentration adjusted to 3 x 10⁵ conidia mL^-1 with a Newbauer haemocytometer.

The antagonist Epicoccum sp. was grown in Petri dishes containing PDA and incubated for 25 days at room temperature. The conidial suspension was prepared by flooding the plates with sterile distilled water and scraping mycelial fragments and conidia with a paint brush. The suspension obtained was filtered through cheesecloth and the final conidial suspension was adjusted to the desired concentrations.

Inhibition of conidial germination and appressorial formation

Germination of conidia and formation of appressoria were induced on the hydrophobic side of parafilm (Filippi, 2004). The parafilm, previously surface sterilized with sodium hypochloride followed by 70% ethanol, was placed on microscopic slides in each Petri dish containing two layers of moistend filter paper to maintain high humidity. A standard solution containing 80 mg of crude extract was diluted in sterile water to obtain six different concentrations (500, 2000, 4000, 6000 and 8000 ppm), in addition to a water control. Twenty microliters of conidia (1 x 10⁵) and crude extract of Epicoccum sp. was dispensed on the hydrophobic surface of the parafilm. The bioassay was run as a completely randomized design with four replications. Conidial germination was assessed after three and six hours and appressorial formation after 12 and 20 hours of incubation at 25 ^oC. The percentage of germinated conidia and formed appressorium were determined by microscopic examination of 100 conidia per replicate.

Relative efficiency of conidial suspension and crude extract of Epicoccum sp. in suppressing rice leaf blast

Planting

The experiment was conducted using cultivar Primavera in plastic trays (15 x 30 x 10 cm) containing 3 kg of soil fertilized with NPK (5 g of 5-30-15 NPK mix + 1 g of Zn and 3 g of ammonium sulfate at planting day). The seeds were sown in eight 10 cm-rows per tray and thinned to 10 to 12 plants per row after germination. Top dressing was done 20 days after seeding with 2 g of ammonium sulfate per tray. The bioassay was run as a completely randomized design with three replications.

Treatments

The experiment was designed to compare rice leaf blast suppression by conidial suspension and crude extract, applied 48 hours before spraying the plants with challenger or sprayed as a mixture of Epicoccum sp. (EP06) and M. oryzae (M.o.). The different concentrations were based on previous exploratory experiments which gave most promising results in the suppression of leaf blast. The following treatments were included: T1 = Crude extract of EP06 (2000 ppm) applied 48 hours before challenge inoculation; T2 = Crude extract of EP06 (4000 ppm) applied 48 hours before challenge inoculation; T3 = Conidial suspension of EP06 (5 x 10⁵ con mL^-1) applied 48 hours before challenge inoculation; T4 = Conidial suspension of a mixture of EP06 (10 x 10⁵ con mL^-1) and M.o. (3 x 10⁵ con mL^-1); T5 = Conidial suspension of a mixture of EP06 (10 x 10¹ con mL^-1) and M.o. (3 x 10⁵ con mL^-1); T6 = Crude extract of EP06 (2000 ppm) mixed with conidia of M.o. (3 x 10⁵ con mL^-1); T7 = Crude extract of EP06 (4000 ppm) mixed with conidia of M.o. (3 x 10⁵ con mL^-1); T8 = Control (M.o., 3 x 10⁵ con mL^-1).

Twenty one-day old plants were inoculated with conidial suspension of M. oryzae, according to the method of Filippi and Prabhu (2001). Following inoculation, the plants were incubated in plastic moist chambers for 24 hours and transferred to greenhouse benches at 27- 30ºC and high humidity until disease evaluation.

Rice leaf blast evaluations were made starting from the appearance of the first lesion using a percentage disease scale according to Notteghem (1981). The disease severity assessed at 2-day intervals was based on 24 rice plants per trial for AUDPC determination.

Induction of disease resistance by crude extract of Epicoccum sp. and enzymatic activity

Induction of disease resistance

Another greenhouse experiment was conducted to study the enzymatic activity and induced resistance by crude extract of Epicoccum sp. (4000 ppm) sprayed 48 hours before the application of challenger M. oryzae. The concentration and application timing were based on significant leaf blast suppression obtained in the previous experiment. Planting, preparation of inoculum of M. oryzae and crude extract of Epicoccum sp. were done as described above. The cultivar Primavera was planted in three plastic trays each of the following treatments: T1 = Crude extract of Epicoccum sp.; T2 = Crude extract of Epicoccum sp. sprayed 48 hours before M. oryzae and T3 = M. oryzae. The inoculation and evaluation was done as described above.

Enzymatic activity

Enzymatic activities of peroxidase, PAL and PR proteins (β-1,3-glucanase and chitinase) were quantified. The sample for analysis was based on the third leaf of 15 rice plants for each treatment. The first collection was done before the inoculation with challenger whereas the second, third, fourth, fifth, sixth and seventh were performed at 24, 48, 72, 96, 120 and 144 hours after inoculation of M. oryzae, respectively. The tests were performed in triplicates. One active enzymatic unit (1.0 U) was defined as variation of absorbance per hour at 25ºC and pH 9.0. The specific activity (U mg^-1) was calculated as the ratio between enzymatic activity previously defined in units (U) and protein content quantified in each sample (mg). Activity was expressed in units (U) mg^-1 protein. For PR proteins, one U was defined as the enzyme activity catalyzing the formation of reducing sugar that increases the absorbancy by 1 unit per hour.

Protein extraction: A sample of five leaves was ground to a fine powder in liquid nitrogen. The buffer solution was composed of 10 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 2 mM DTT, 1 mM PMSF, leptin (10 µg mL^-1) and aprotinin (10 µg mL^-1). Protein determination was according to Bradford (1976).

β-1,3-glucanase activity (EC 3.2.1.6): Activity of β-1,3-glucanase was assayed by measuring the rate of reducing sugar production using laminarin (Sigma) as the substrate (Pan et al., 1991). DNS reagent was used as the colorimetric agent.

Chitinase activity (EC 3.2.1.14): Chitinase activity was assayed by a modified method of Pan et al. (1991). The rate of N-acetyl glucosamine production was measured using colloidal chitin as the substrate. The 3,5-dinitrosalicylic acid (DNS) reagent was used as the colorimetric agent.

Peroxidase activity (EC 1.11.1.7): Peroxidase activity was assayed by measuring the rate of 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) oxidation, using its own colorimetric property. One unit was defined as the enzyme activity catalyzing the formation of 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) that increases the absorbancy by 1 unit per hour (Keesey, 1987).

Phenylalanine ammonia-lyase (EC 4.3.1.5): The determination of PAL activity was performed utilizing 2 mL of a 10 mM phenyalanine suspension in 0.1 M borate buffer pH 9.0, and 50 µL of plant protein extract. The mixture was homogenized and submitted for quantification of the product in spectrophotometer (Fento 600 Plus) at 290 nm.

Scanning electron microscopy

Leaves of rice plants of all three treatments were collected 24 hours after inoculation of challenger M. oryzae for scanning electron micrograph (SEM). The leaf bits were placed in microtubes containing Karnovsky fixative (glutaraldehyde and 2% isofar in combination with 2% formaldehyde), and later in cacodylate buffer (pH 7.2 plus sucrose 3%) for 24 hours at 23°C. Later, the samples were washed three times in 0.1M cacodylate buffer for 10 minutes each time, dehydrated in an ethanol series (70, 80, 90 and 99.1%), and submitted to critical point drying with liquid CO₂ (Autosamdri 815) during 40 minutes. The dry specimen was mounted on stubs using double-sided adhesive tape. The stubs were aluminum discs of 2.5 cm diameter and 0.8 cm thick which were previously sterilized with acetone and isopropylene alcohol. Later, the stubs were submitted for two minutes for the deposition of ultrathin films of gold using Denton Vaccum, Desc VA. After sputter-coated with gold, these stubs containing the samples were taken for observation in a Jeol JSM-6610 scanning electron microscope equipped with EDS.

Statistical analysis

The data on antagonism of Epicoccum sp. against rice pathogens, suppression of leaf blast by conidia of Epicoccum sp., conidial germination and formation of appressoria by crude extract of Epicoccum sp., induction of resistance by crude extract of Epicoccum sp. and enzymatic activity in plants challenged with the pathogen were submitted to analysis of variance using the Statistical Package for the Social Sciences (SPSS), version 18.0. Means were compared by Duncan's test (P<0.05). Leaf blast severity data in percentages were transformed to arc sine √x+0.5.

The lethal dose for conidial germination expressed in relation to the concentration of crude extract (ppm) that caused 50% inhibition (LD₅₀) was determined. The method of probit-log dosage analysis according to Finney (1975) was utilized for determining the LD₅₀ assuming that the tolerance of conidia to concentrations of crude extract has a normal distribution. The method consists in estimating the linear regression of the probit value, corresponding to the percentage inhibition of conidia on log concentration of crude extract. The percentage conidial inhibition was obtained in relation to the germination of the control treatment.

RESULTS

Antagonism of Epicoccum sp. against rice pathogens

All rice pathogens tested using dual culture with Epicoccum sp. showed reduction in mycelial growth in relation to their respective controls. They also showed significant differences among the test pathogens in relation to inhibition percentage (Figure 1). The antagonism of Epicoccum sp. against M. oryzae was 42.50%.

Inhibition of conidial germination and appressorial formation of M. oryzae by crude extract of Epicoccum sp.

The percentage of germination and appressorial formation were significantly reduced in all six concentrations of crude extract in relation to control. The probit-log dosage curves for conidial germination are presented in Figure 2. The LD₅₀ values were 393.0 and 326.6 ppm for 6 and 12 hours, respectively. The high percentage of conidial germination inhibition even after 6 hours shows that the crude extract contains a potential anti-fungal agent. The germinated conidia that formed appressoria at 12 and 20 hours after induction were reduced by 97.7% and 95.68%, respectively, with a crude extract concentration of 6000 ppm, compared with the control (Figure 3A, B).

Relative efficiency of conidial suspension and crude extract of Epicoccum sp. in the suppression of leaf blast

The results of the experiment are presented in Table 1. Treatment differences were significant in relation to leaf blast severity as well as AUDPC compared with the control. The crude extract was as efficient as conidial suspension in reducing leaf blast.

In relation to timing of application, conidial suspension or crude extract applied 48 hours before the application of challenger M. oryzae suppressed leaf blast. There was, however, no statistical difference between the application 48 hours before the challenger and simultaneous application as a mixture. The application of crude extract at 4000 ppm, 48 hours before the application of M. oryzae showed AUDPC of 1.1 compared to 45.0 in the control, which corresponds to 97.6% reduction in leaf blast.

Based on these results, the concentration of 4000 ppm of crude extract of Epicoccum sp. and the application timing of 48 hours before spraying the challenger M. oryzae were utilized in further studies.

Induction of resistance by crude extract of Epicoccum sp., enzymatic activity and scanning electron microscopy

The application of crude extract of Epicoccum (4000 ppm) in rice plants 48 hours before spraying the challenger (M.o.) reduced leaf blast severity assessed at 2-day interval up to 9 days, in relation to plants sprayed only with M. oryzae. No disease symptoms were evident in plants sprayed with Epicoccum sp. (Figure 4).

Spraying rice plants with crude extract of Epicoccum sp. significantly increased the activity of PAL and chitinase 96 hours after the application of challenger. PAL activity increased in plants challenged as well as induced and challenged 48 and 72 hours after inoculation with M.o.. Chitinase activity increased only 72 hours after challenge inoculation. Chitinase activity in plants sprayed with M. oryzae, Epicoccum sp. and resistance induced plants did not differ but differed significantly compared to the control sprayed with water (Figure 5A, C).

The activities of peroxidase and β-1,3-glucanase increased in plants sprayed with crude extract of Epicoccum sp., 24 hours after the application of challenger (Figure 5B, D), the critical period in the infection process of M.o Peroxidase activity increased 48 and 72 hours after induced resistance followed by challenge. After 72 and 96 hours of inoculation with M.o. all treatments differed from the control in relation to peroxidase activity (Figure 5B). β-1,3-glucanase activity significantly increased in plants that received only crude extract of Epicoccum sp., 24 after inoculation of the challenger (Figure 5D).

In leaves treated with only crude extract of Epicoccum sp. the presence of crystal fragments of Epicoccum sp. can be observed without the presence of M.o. (Figure 6A) whereas in leaves inoculated with crude extract of Epicoccum sp. followed by M. oryzae the conidia of M. oryzae with deformed appressoria were evident (Figure 6B). In control inoculated only with the pathogen, the conidia and appressoria were formed normally without any deformation (Figure 6C).

DISCUSSION

Epicoccum spp. is considered a saprophytic fungus and phylloplane inhabitant in rice (Araújo et al., 2010). In the present study, the reduced mycelial growth and significant colony inhibition of M. oryzae, R. solani, S. oryzae, M. albescens and C. miyabeanus indicate its potential to control several rice diseases. These results are in agreement with the earlier reports of mycelial growth inhibition by Epicoccum sp. of various pathogens such as M. oryzae (Araújo et al., 2010), Colletotrichum gloesporioides (Pandey et al., 2007), Rhizoctonia solani (Lahlali & Hijri, 2010), Macrophomina phaseolina (Hashem, 2004) and Fusarium sambuciunum (El-Kot, 2008).

The crude extract is being widely utilized with the objective of identifying potential biological agents. Li et al. (2011) showed that the crude extract of Streptomyces globisporus inhibited mycelial growth, conidial germination and appressorial formation of M. oryzae in histological observations of cut leaves. According to Madrigal et al. (1991) the secondary metabolite flavipine poduced by Epicoccum sp. inhibited conidial germination of several plant pathogens. In the present study, the partially purified crude extract of Epicoccum sp. affected conidial germination and appressorial formation in all six concentrations tested. The reduction in appressorial formation was as high as 97.7% and is greater than the inhibition of conidial germination. Further studies are underway with reference to components of crude extract that are responsible for antagonistic effect against M. oryzae.

In this study, the antagonistic effect of Epicoccum sp. detected in vitro was also confirmed in vivo in greenhouse inoculation tests. An experiment was conducted to determine the relative efficiency of crude extract and conidial suspension in suppressing leaf balst. The results showed that crude extract of Epicoccum sp. is as efficient as spraying conidial suspension of the same (Table 1). The application of crude extract of Epicoccum sp. (2000 and 4000 ppm) sprayed 48 hours before the inoculation with challenger M. oryzae reduced leaf blast severity by 97.56% in relation to control. The crude extract applied as a mixture with the conidial suspension of M. oryzae or as a mixture of conidia of both fungi also controlled leaf blast compared with control. The efficiency of conidial suspension of Epicoccum sp. in the suppression of other plant diseases and its potential as biological agent were reported by several investigators (Madrigal et al., 1991; Wittig et al., 1997; Pieckenstain et al., 2001; Hashem, 2004; Pandey et al., 2007; El-Kot, 2008, Koutb & Ali, 2010; Lahlali & Hijri, 2010; Musetti et al., 2011).

The Epicoccm sp. isolate utilized in the present study (EP06) was obtained from the rice phylloplane. Kawamata et al. (2004) verified that 7% of 967 rice phylloplane fungal isolates reduced leaf blast severity, including Epicoccum sp. The chances of success of phylloplane constituent as biological agent are considered greater, because they already developed the potential to colonize their host plants.

The results in the present study demonstrated the efficiency of crude extract in controlling leaf blast under greenhouse conditions. The hypothesis that crude extract of Epicoccum sp. activating the plant defense mechanisms is supported by our results. It is possible that PAL and chitinase may not have participated in the initial defense which is important for successful infection. PAL is a precursor and regulator of salicylic acid, the plant hormone required for induction of SAR (Chen et al., 2009; Dempsey et al., 2011). The results indicate that crude extract of Epicoccum sp. did not induce SAR. However, the antagonistic effect of crude extract with M. oryzae may have promoted the liberation of molecules that had effect on PAL and chitinase (PR-3, PR-4, PR-8 and PR-11), which was observed 96 hours after challenge inoculation with the pathogen. It is known that the Pal 1 gene that codifies PAL is activated by jasmonic acid and ethylene during the process of plant defense (Shoresh et al., 2010).

On the other hand, the crude extract of Epicoccum sp. induced increased activity of peroxidase and β-1,3-glucanase (PR-2). In earlier in vitro tests the production of chitinase and β-1,3-glucanase were not detected by the isolate of Epicoccum sp. (data not shown). However, we need further studies concerning enzyme production by Epicoccum sp. and composition of its crude extract. The increased enzymatic activity refers to its proper plant product and known to be related to the activation of ISR. Increased activity of β-1,3-glucanase and peroxidase during leaf blast suppression by rhizobacteria and acibenzolar-S-metyl was reported by Filippi et al. (2011) and Côrtes et al. (2008). Peroxidase is related to diverse responses to plant stress. In the present study, as the plants were not subjected to abiotic stress, we may conclude that the crude extract is responsible for increased enzymatic activity, mainly after 24 hours, the critical period for the suppression of infection in which the fungus initiates the penetration process. These results are further supported by our SEM analysis, confirming the validity of data on suppression of rice leaf blast severity compared with the control.

According to Walters et al. (2007) and Pieterse et al. (2009), three types of systemic resistance ae characterized which are effective against biotrophic and necrotrophic plant pathogens: SAR, ISR, and resistance induced by β-aminobutíric acid (ABA-IR). The metabolic pathways that break or control these systems and the associated defense responses were well characterized in Arabidopsis thaliana (Van der Ent et al., 2008; Pieterse et al., 2009). SAR, depending on the presence of salicylic acid, can be induced by necrotizing pathogens, and by specific elicitors which are associated to the related pathogenesis proteins and regulated by proteins and by NPR1. ISR can be induced by beneficial microorganisms such as rhizobacteria and the fungus Trichoderma spp. which do not require the presence of salicylic acid and not PR dependent, but dependent on jasmonic acid, ethylene and of NPR1. On the other hand, the ABA-IR metabolic pathway depends on the presence of salicylic acid and abscisic acid. The results herein presented allow us to conclude that Epicoccum sp. as well as its crude extract elicited plant defense mechanisms in rice, but what is still lacking is the knowledge on the metabolic pathway that is activated. For this it will be necessary to quantify plant hormones, jasmonic acid, ethylene and salicylic acid to explain when and which pathways are elicited by its product. Also, further studies are necessary to elucidate the fraction of crude extract that has the capacity to elicit the plant defense and establish the antibiosis with the rice pathogen. In addition, through molecular techniques it is possible to clone genes related to defense pathways of host plants such as elicitors by crude extract of Epicoccum sp., as well as fractions of crude extract responsible for antibiosis against rice pathogens. Various investigators provided evidence regarding the induction of SAR in rice after the application of microorganisms utilizing avirulent isolates of M. oryzae and Bipolaris sorokiniana (Smith & Métraux 1991; Manandhar et al., 1998; Tsukamoto et al., 1999; Ashizawa et al., 2005; Filippi et al., 2007) as well as ISR in rice with Pseudomonas fluorescens for the control of sheath blight (Nandakumar et al., 2000; Radjacommare et al., 2004), with rhizobacteria and with Bacillus sp. for leaf blast (Nascimento, 2009; Filippi et al., 2011). Umamaheswari et al. (2009) showed that seed treatment and foliar application of biocontrol agents induced resistance to Alternaria alternata in watermelon. Besides, the six pre-treatment agents increased protection of the plant to the pathogen as shown by the increased activity of PAL, peroxidase and β-1,3-glucanase.

Our SEM results indicate that the increased activity of these products is capable of inhibiting germination and appressorial formation. The crude extract did not impair the initial phases of infection because the conidia of M. oryzae are visible, germinated, and formed appressoria, however, without success for penetration.

There are still some limitations in adopting resistance induction (RI) in commercial crops, including instability of the efficiency of some inducers, growers' demands in relation to quality and performance of the elicitor. Under field conditions, the expression of RI is influenced by climatic conditions, genotype and nutritional status. Elicitors such as crude extract of Epicoccum spp., which possess both characteristics of antagonism and resistance induction, are highly promising for integrated management of cultivated plants.

The results of this study showed that Epicoccum sp., obtained from the phylloplane of rice plants in commercial fields, showed antagonistic potential against M. oryzae, R. solani, S. oryzae, M. albescens and C. miyabeanus. Also, the partially purified crude extract of Epicoccum sp. as well as conidial suspension reduced conidial germination, appressorial formation, rice leaf blast severity and induced plant defense responses by increasing activity of peroxidase and β-1,3-glucanase, 24 hours after the application of challenger M. oryzae. These results further confirm that this fungus is both antagonist and systemic resistance inducer against M. oryzae. Further studies are necessary with crude extract of Epicoccum sp. for identifying the elicitor molecule(s) during the initial stages of the rice-M. oryzae interaction responsible for induction of ISR, as well as studies on transduction of signals used for activating local and systemic defense.

ACKNOWLEDGEMENTS

We thank Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG and Universidade Federal de Goiás - UFG for providing financial support for this research, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES for financial support to the first author, and the Multiuse Microscopy Laboratory (LabMic) of UFG for scanning electron microscopic analysis.

Received 28 March 2013

Accepted 23 April 2013

TPP 2013-0044 -

Section Editor: Bernardo A. Halfeld-Vieira

• Araújo LG, Mendanha RA, Gonçalves FJ, Fillipi MC, Silva GB, Prabhu AS (2010) Antibiose de fungos do filoplano de plantas de arroz a Magnaporthe oryzae. Revista de Biologia Neotropical 7:1-6.
• Ashizawa T, Zenbayashi K, Sonoda R (2005) Effects of preinoculation with an avirulent isolate of Pyricularia grisea on infection and development of leaf blast lesions caused by virulent isolates on near-isogenic lines of Sasanishiki rice. Journal of General Plant Pathology 71:345-350.
• Barnett HL, Hunter BB (2006) Illustrated Genera of Imperfect Fungi. 4^th Ed. Saint Paul MN, USA. APS Press.
• Beauverie F (1901) Essais d'immunisation des végétaux contre les maladies cryyptogamiques. Comptes Rendus des Seances de l'Academie des Sciences 133:307.
• Bradford M (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72:248-254.
• Chen Z, Zheng Z, Huang J, Lai Z, Fan B (2009) Biosynthesis of salisylic acid in plants. Plant Signaling & Behavior 4:493-496.
• Côrtes MVCB, Viana HF, Silva FR, Lobo VLS, Silva GB, Prabhu AS, Filippi MCCF (2008) Quantificação da atividade enzimática de proteínas relacionadas à patogênese no patossistema Oryza sativa/Magnaporthe grisea Boletim de Pesquisa e Desenvolvimento 34. Santo Antônio de Goiás GO, Brazil. Embrapa Arroz e Feijão.
• De Cal MA, Larena I, Liñam M, Torre R, Lamarca N, Usall J, Domenichini P, Bellini A, Eribi XO, Melgarejo P (2009) Population dynamics of Epicoccum nigrum, a biocontrol agente against brown rot in stone fruit. Journal of Applied Microbiology 106:592-605.
• Dempsey D'MA, Vlot AC, Wildermuth MC, Klessig DF (2011) Salicylic acid biosynthesis and metabolism. The Arabidopsis Book 9:3-24.
• Derbalah AS, El Kot GAN, Hamza AM (2011) Control of powdery mildew in okra using cultural filtrates of certain bio-agents alone and mixed with penconazole. Archives of Phytopathology and Plant Protection 44:2012-2023.
• Doohan F (2005) Fungal pathogens of plants. In: Kavanagh K (Ed.) Fungi: biology and applications. Chichester UK. John Wiley. pp. 232-263.
• El-Kot GAN (2008) Biological control of black scurf and dry rot of potato. Egyptian Journal of Phytopathology 36:45-56.
• Falconi CJ, Mendgen K (1994) Epiphytic fungi on apple leaves and their value for control of the postharvest pathogens Botrytis cinerea, Monilinia fructigena and Penicillium expansum Journal of Plant Disease and Protection 101:38 - 47.
• FAO (2011) Situación alimentaria mundial. Available at: www.fao.org.br. Accessed on May 7, 2011.
• Filippi MCC (2004) Signalling pathway in appressorium formation in Magnaporthe grisea PhD Thesis, Texas A & M University. College Station TX, USA.
• Filippi MCC, Prabhu AS (2001) Phenotypic virulence analysis of Pyricularia grisea isolates from Brazilian upland rice cultivars. Pesquisa Agropecuária Brasileira 36:27-35.
• Filippi MCC, Silva GB, Prabhu AS (2007) Indução de resistência à brusone em folhas de arroz por isolado avirulento de Magnaporthe oryzae Fitopatologia Brasileira 32:387-392.
• Filippi MCC, Silva GB, Silva-Lobo VL, Cortes MVCB, Moraes AJG, Prabhu AS (2011) Leaf blast (Magnaporthe oryzae) suppression and growth promotion by rhizobacteria on aerobic rice in Brazil. Biological Control 58:160-166.
• Finney DJ (1975) Statistical Methods in Biological Assay. London UK. Griffin.
• Fisher MC, Henk DA, Briggs CJ, Brownstein JS, Madoff LC, Mccraw SL, Gurr SJ (2012) Emerging fungal threats to animal, plant and ecosystem health. Nature 484:186-194.
• Hashem M (2004) Biological control of two phytopathogenic fungal species isolated from the rhizoplane of soybean (Glycine max). Czech Mycology 56:223-238.
• Hashem M, Ali EH (2004) Epicoccum nigrum as biocontrol agent of Pythium damping-off and root rot of cotton seedlings. Archives of Phytopathology and Plant Protection 37:283-297.
• Huang HC, Bremer E, Hynes RH, Erickson RS (2000) Foliar application of fungal biocontrol agents for the control of white mold of dry bean caused by Sclerotinia sclerotiorum Biological Control 18:270-276.
• Kawamata H, Narisawa K, Hashiba T (2004) Suppression of rice blast by phylloplane fungi isolated from rice plants. Journal of General Plant Pathology 70:131-138.
• Keesey J (1987) Biochemica Information. Indianapolis IN, USA. Boehringer Manhein Biochemicals.
• Kortekamp A (1997) Epicoccum nigrum Link: A biological control agent of Plasmopara viticola (Berk. et Curt.) Berl. et De Toni. Vitis 36:215-216.
• Koutb M, Ali EH (2010) Potential of Epicoccum purpurascens Strain 5615 AUMC as a biocontrol agent of Pythium irregulare root rot in three leguminous plants. Mycobiology 38:286-294.
• Lahlali R, Hijri M (2010) Screening, identification and evaluation of potential biocontrol fungal endophytes against Rhizoctonia solani AG3 on potato plants. FEMS Microbiology Letters 311:152-159.
• Li Q, Jiang Y, Ning P, Zheng L, Huang J, Li G, Jiang D, Hsiang T (2011) Suppression of Magnaporthe oryzae by culture filtrates of Streptomyces globisporus JK-1. Biological Control 58:139-148.
• Madrigal C, Tadeo JL, Melgarejo P (1991) Relationship between flavipin production by Epicoccum nigrum and antagonism against Monilinia laxa Mycological Research 95:1375-1381.
• Manandhar HK, Jorgensen HJL, Mathur SB, Smedegaard-Petersen V (1998) Resistance to rice blast induced by ferric chloride, di-potassium hydrogen phosphate and salicylic acid. Crop Protection 17:323-329.
• McSpadden-Gardener B, Fravel DR (2002) Biological control of plant pathogens: Research commercialization, and application in the USA. Plant Health Progress. Avaliable at: www.apsnet.org/publications/apsnetfeatures/Pages/biocontrol.aspx. Accessed on May 25, 2012.
• Musetti R, Grisan S, Polizzotto R, Martini M, Paduano C, Osler R (2011) Interactions between 'Candidatus Phytoplasma mali' and the apple endophyte Epicoccum nigrum in Catharanthus roseus plants. Journal of Applied Microbiology 110:746-756.
• Nandakumar R, Babu S, Viswanathan R, Raguchander T, Samiyappan R (2000) Induction of systemic resistance in rice against sheath blight disease by Pseudomonas fuorescens Soil Biology and Biochemistry 33:603-612.
• Nascimento IO (2009) Isolamento, identificação e seleção de Bacillus sp. para o biocontrole de fitopatógenos do arroz. MSc Dissertation, Universidade Estadual do Maranhão. São Luís MA, Brazil.
• Notteghem JL (1981) Cooperative experiment on horizontal resistance to rice blast. In: BLAST and upland rice: Report and recommendations from the meeting for international collaboration in upland rice improvement. Los Baños The Philippines. International Rice Research Institute. pp. 43-51.
• Pal KK, McSpadden-Gardener BB (2006) Biological control of plant pathogens. Available at: www.apsnet.org/edcenter/advanced/topics/Pages/BiologicalControl.aspx. Accessed on May 25, 2012.
• Pan SQ, Ye XS, Kuc J (1991) Association of a β-1,3-glucanase activity and isoform pattern with systemic resistance to blue mold in tobacco induced by stem injection with Peronospora tabacina or leaf inoculation with tobacco mosaic virus. Physiological and Molecular Plant Pathology 39:25-39.
• Pandey RR, Sharma G, Tripathi SK, Singh AK (2007) Litterfall, litter decomposition and nutrient dynamics in a subtropical natural oak forest and managed plantation in northeastern India. Forest Ecology and Management 240:96-106.
• Pieckenstain FL, Bazzalo ME, Roberts AMI, Ugalde RA (2001) Epicoccum purpurascens for biocontrol of Sclerotinia head rot of sunflower. Mycological Research 105:77-84.
• Pieterse CMJ, Leon-Reyes A, Van Der Ent S, Van Wees SCM (2009) Networking by small-molecule hormones in plant immunity. Nature Chemical Biology 5:308-316.
• Prabhu AS, Filippi MC, Silva GB, Lobo VLS, Moraes OP (2009) An unprecedented outbreak of rice blast on a newly released cultivar BRS Colosso in Brazil. In: Wang GL, Valente B (Eds.) Advances in Genetics, Genomics and Control of Rice Blast Disease. New York NY, USA. Springer. pp. 257-266.
• Radjacommare R, Kandan A, Nandakumar R, Samiyappan R (2004) Association of the hydrolytic enzyme chitinase against Rhizoctonia solani in rhizobacteria-treated rice plants. Journal of Phytopathology 152:365-370.
• Ramos LN (2009) Estrutura populacional e parâmetros epidemiológicos de isolados de Magnaporthe grisea (Barr). MSc. Dissertation, Universidade de Brasília. Brasília DF, Brazil.
• Romeiro RS (2007) Controle Biológico de Enfermidades de Plantas. Viçosa MG, Brazil. Editora UFV.
• Sena APA (2012) Supressão de brusone nas folhas em arroz por Epicoccum sp. MSc Dissertation, Universidade Federal de Goiás. Goiânia GO, Brazil.
• Shoresh M, Mastouri F, Harman GE (2010) Induced systemic resistance and plant responses to fungal biocontrol agents. Annual Review of Phytopathology 48:21-43.
• Smith JA, Métraux JP (1991) Pseudomonas syringe pv. syringae induces systemic resistance to Pyricularia oryzae in rice. Physiological and Molecular Plant Pathology 39:451-461.
• Tsukamoto H, Tsutsumi F, Onodera K, Yamada M, Fujimori T (1999) Biological control of rice leaf blast with Exserohilum monoceras, a pathogen of Echinochloa species. Annals of the Phytopathological Society of Japan 65:543-548.
• Umamaheswari C, Sankaralingam A, Nallathambi P (2009) Induced systemic resistence in watermelon by biocontrol agents against Alternaria alternate. Archives of Phytopathology and Plant Protection 42:1187-1195.
• Van Der Ent S, Ton J, Koornneef A, Pieterse CMJ (2008) Induced resistance - Orchestrating defence mechanisms through cross-talk and priming. Annual Plant Reviews 34:334-370.
• Van Loon LC, Pieterse CMJ (2006) Significance of inducible defense-related proteins in infected plants. Annual Review of Phytopathology 44:35-62.
• Walters D, Newton A, Lyon G (2007) Induced Resistance for Plant Defense. A sustainable Approach to Crop Protection. Oxford UK. Blackwell.
• Wei G, Kloepper JW, Tuzun S (1991) Induction of systemic resistance of cucumber to Colletotrichum orbiculare by select strains of plant growth-promoting rhizobacteria. Phytopathology 81:1508-1512.
• Wittig HPP, Johnson KB, Pscheidt JW (1997) Effect of epiphytic fungi on brown rot blossom blight and latent infections in sweet cherry. Plant Disease 81:383-387.
• Yoshikawa M, Keen NT, Wang MC (1983) A receptor on soybean membranes for a fungal elicitor of phytoalexin accumulation. Plant Physiology 73:497-506.

Publication Dates

• Publication in this collection
  27 Aug 2013
• Date of issue
  Oct 2013

History

• Received
  28 Mar 2013
• Accepted
  23 Apr 2013